Peptides for enhancing resistance to microbial infections

ABSTRACT

The present invention is related to novel bioactive pentapeptides, pentarphins, the main indication of which is enhancing phagocytic activity of macrophages against microbes. In particular, the cyclopentapeptide, cyclo(Val-Lys-Gly-Phe-Tyr), termed cyclopeptarphin, was 100 times more active than tuftsin. Cyclopentarphin was non-toxic even at concentrations 1000 times higher than the minimum active dose, while being non-immunogenic. Furthermore, cyclopentarphin is more stable to enzymatic cleavage in vitro as compared to linear pentarphin and tuftsin and, hence, its life span in vivo is also larger than that of linear peptides. High efficacy and safety of cyclopentarphin enable elaboration of novel drugs that enhance the resistance of human and animal organisms to microbes and micro particles.

This is national stage application under 35 U.S.C. section 371 ofinternational application WO 03/061683 filed on Jan. 23, 2003 andpublished on Jul. 31, 2003, said international application claimingpriority of the Finnish national patent application FI20020000121 filedon Jan. 23, 2002.

FIELD OF INVENTION

The present invention is related to human and animal medicine, and morespecifically, to improvement of the resistance of humans and animals tomicrobial infections and/or enhancement of the therapeutic effect ofantibiotics. According to this invention, certain natural proteinscontain peptide segments that can augment activity of macrophages andand other cells of immune system. These peptides can be used for thecreation of novel drugs.

BACKGROUND OF INVENTION

A specific enzyme, leucokinase, that is located in the outer membrane ofthe neutrophils, spilits leucocinin, a leucophilic fraction ofimmunoglobulin G (IgG) and produces a phagocytosis-stimulatingtetrapeptide (Najjar V. A. and Nishioka K., 1970, Nature 228, pp.672-673). That tetrapeptide was subsequently named “tuftsin” (Najjar V.A. and Nishioka K., 1970, Nature 228, pp. 672-673; Sieminon I. Z. andKluczyk A., 1999, Peptides 20, pp. 645-674). Tuftsin (Thr-Lys-Pro-Arg)is a 289-292 sequence in the C_(H)2 domain of the Fc subunit of humanIgG1 heavy (H) chain. It was originally found that tuftsin stimulatesphagocytosis after binding to polymorphonuclear cells (ConstantopoulosA. and Najjar V. A., 1972, Cyobios. 6, pp. 97-100; Najjar V. A. andConstantopoulos A. A., 1972, J. Reticulendothel. Soc. 12, pp. 197-215;Najjar V. A., 1979, Clin. Wochenschr. 57, pp. 751-756; Najjar V. A.,1980, Adv. Exp. Med. Biol. 121 A, 131-147; Najjar V. A., 1983, Ann. NYAcad. Sci. 419, pp. 1-11). Subsequently, tuftsin was also shown tostimulate the phagocytosis activity of monocytes-macrophages (Coleman D.L., 1986, Eur. J. Clin. Microbiol. 5, pp. 1-5). Potentially, tuftsincould be used as a drug component to increase phagocytosis activities.However, the drawback is that tuftsin activity is very low demanding itshigh concentrations in blood circulation. Another drawback is thathalf-lives of linear peptides in blood are short.

In 1980 the existence of a β-endorphin-like sequence in C_(H)3 domain ofthe Fc subunit of human IgG1-4H-chain was reported (Julliard J. H. etal., 1980, Science 208, pp. 183-185). To isolate ACTH and β-endorphinfrom human placenta, the authors used immobilized antibodies to thesehormones as affinity absorbents. A 50 kDa protein was thereby isolatedand found to be an H-chain of IgG. Elucidation of the origin of such aneffect led to the discovery of ACTH- and β-endorphin-like sequences inthe H-chain. It was found that the human IgG1 H-chain fragment 364-377(SLTCLVKGFYPSDI (SEQ ID NO:1); see FIG. 1) was 40% homologous toβendorphin fragment 10-23 (SQTPLVTLFKNAII (SEQ ID NO:2)). An artificialpeptide (14 amino acid residues) corresponding to the β-endorphin-likehuman IgG1 sequence was synthesized and found to interact with rat brainreceptors for β-endorphin (Houck J. C. et al., 1980, Science 207,78-80). Our group synthesized a decapeptide SLTCLVKGFY(SEQ ID NO:3)(termed immunorphin) corresponding to the human IgG1 H-chain sequence364-37. It was demonstrated to compete with [¹²⁵I]β-endorphin forhigh-affinity receptors on murine peritoneal macrophages (K_(i)=2.5 nM;Zav'yalov V. P. et al., 1996, Immun. Lett. 49, 21-26). Later on it wasalso demonstrated to compete with [¹²⁵I]β-endorphin for high-affinityreceptors on T lymphocytes from the blood of healthy donors (K_(i)=0.6nM). Tests of the specificity of the receptors revealed that they areinsensitive to an antagonist of opioid receptors naloxone and[Met⁵]enkephalin, i.e. they are non-opioid receptors. The displacementassays demonstrated that pentapeptide VKGFY(SEQ ID NO:4), termedhereinafter as pentarphin, was the shortest immunorphin fragment,capable of inhibiting [¹²⁵I]β-endorphin binding to non-opioid receptorson murine macrophages (K_(i)=12 nM) and human T lymphocytes (K_(i)=15nM). According to the present invention, the primary effect ofpentarphin, following binding to specific cell surface receptors,consists of the stimulation of the functions of macrophages and Tlymphocytes.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1. Comparison of amino acid sequences of [Met⁵]enkephalin (SEQ IDNO:11), β-endorphin (SEQ ID NO:10), β-endorphin-like fragments of humanimmunoglobulin GI (HuIgG1) heavy (H) chain (SEQ ID NO:1), immunorphin(SEQ ID NO:3), and pentarphin (SEQ ID NO:4).

FIG. 2. Structure of cyclopentarphin.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is related to novel bioactivecompounds—pentapeptide Val-Lys-Gly-Phe-Tyr (SEQ ID NO:4, and its cyclicanalog—cyclo(Val-Lys-Gly-Phe-Tyr), capable of enhancing phagocyticactivity of mouse peritoneal macrophages against virulent bacterialstrains and of binding to a variety of cells of immune system. The termpentarphin is used hereafter for the linear sequence described above. Ina larger meaning the term pentarphins is used for the linear or cyclicsequences or for sequences containing mentioned sequences within theirmolecule and having related biological activities as the linear orcyclic pentarphin.

According to this invention, during phagocytosis pentarphins bind to thespecific high-affinity receptors on macrophage surface and enhance thecapacity of phagocytic cells to digest captured microbes. However, forexample, a virulent bacterium Salmonella typhimurium has adapted, asmany other virulent microorganisms, in the process of natural selection,to protect themselves against the bactericidal action of phagocytes.Such adapted microbes release in the surrounding medium so-called“virulence factors” that interfere with the process of formation of ajunction between phagosomes (bubbles formed by cell membrane thatcontain captured microbes) and lysosomes. As a result, phagolysosomesare not formed and lysosomal enzymes have no access to microbes andtherefore cannot digest them. Data presented in Table 1 show that in theabsence of pentarphins (control) captured microorganisms were notdigested but, in contrast, propagated themselves inside phagocytes (PNincreases from 10.17±0.18 to 15.50±0.34 between 2 and 7 h ofphagocytosis). Pentarphin and cyclopentarphin do not influence viabilityand growth of S. typhimurium, that is, they are notpeptides-antibiotics. Similar to tuftsin, both peptides stimulate thecapacity of phagocytes to digest captured microorganisms.

Pentarphins have following advantages as compared to classicalantibiotics:

-   -   1. Unlike antibiotics that act directly on microorganism,        pentarphins affect phagocytes stimulating their digestive        function. Therefore, the activator peptides can be effective in        microbial infections, i.e. to be universal antimicrobial agents.    -   2. Antibiotics are toxic and cause a number of undesirable side        effects (allergy, disbacteriosis, changes in blood cellular        content, impairments of liver, kidney and the central nervous        system functions etc.). Pentarphins are non-toxic, since the        products of their hydrolysis are natural amino acids, while the        only effect is the stimulation of the immune system.

Antibiotics are widely used, commercially important drugs for treatmentof a multitude of infectious diseases of human and higher animals.However, antibiotics have severe drawbacks for their toxicity, narroweffective dose range, and various side effects. Therefore, it ispropitious to try to increase their activities by other means. Highefficacy and safety of pentarphins form the basis for elaboration ofnovel effective therapeutic agents that enhance the resistance of humanand animal organisms to pathogenic microbes or other microparticleseliminated by macrophages. In addition, it appeared promising to workout combined preparations of pentarphins and antibiotics. Thecombinatory drugs influence microbe and macrophage simultaneously. Suchan approach will allow therapeutic doses of antibiotics and consequentlytheir toxicity to be significantly reduced, whereas antimicrobialactivity of preparation as a whole to be significantly increased.

In this invention, it has been shown that certain favourablephysiological effects of antibiotics can be amplified by biologicallyactive peptides, preferably cyclopeptides, corresponding to theβ-endorphin-like sequences of human IgG1-4 subclasses. The mentionedcyclopeptides can be commercially produced by synthetic techniques. Thepresent invention provides definite improvement of drugs based onantibiotics designed for treatment of humans and animals. The peptideparts of such drugs include the biologically active cyclopeptidescorresponding to the β-endorphin-like sequence of IgG subclasses ofdifferent animal species. Although it was impossible to testexperimentally all the potentially bioactive peptide structures, suchstructures were revealed by extensive comparisons of available aminoacid sequences of IgG subclasses from different species by computer andmolecular modeling techniques taking into account the data on thelocalization of the β-endorphin-like sequence of human IgG1-4 subclassesand the experimental data on the competition of pentarphin with[125I]β-endorphin for the common receptors. Therefore the presentinvention is not limited to the linear or cyclic structure ofpentapeptide VKGFY (SEQ ID NO:4) but also includes related structuresfrom other animals with the same biological effects, i.e. the capabilityto enhance the activity of macrophages and/or bind to immunocompetentcells. It is reasonable to assume that in the process of naturalselection the changes in the amino acid sequence of the pentarphin-likesite of IgG were selected not to abolish the biological activities ofthe site. Consequently, all peptides corresponding to this site in allIgG subclasses of different species might reproduce the activity ofpentarphin.

To test a synergy between antibiotics and pentarphins, we employed theclassic macrophage assays with mouse peritoneal macrophages in a cellculture system. The cell culture conditions are strongly reminiscent ofthat of blood circulation. In fact, in cell cultures, which are commonlyused for testing of potential drugs, the conditions are strictlymaintained similar to the blood circulation as to the temperature, pH,buffer, minerals, CO₂- and O₂-partial pressures, and so on. Thus, it ishighly predictable that the biologically active compositions of thepresent invention can be used as medical drugs for humans and animals.Such effective drugs are very desirable for treatment persons with alowered resistance to microbial infections like those having differentkinds of immunodeficiency, for example, AIDS.

It was demonstrated that pentarphins (see Example 4) have very low or notoxicity, because of their amino acid nature. On the other hand, theyare not immunogenic. Therefore they are extremely suitable componentsfor any drug formulations. For certain purposes derivatives ofpentarphins may be useful. Larger pentarphin polymers (repeatedpolypeptide sequences) will possibly be immunogenic. However, suchpolymers or derivatives may possess relatively higher activity, whichcan make their usage favorable.

The role of macrophages is essentially to remove extremely small foreignparticles, for example microbial cells, from the organism. The presentinvention indicates that this process can be accelerated by pentarphins.One would expect that macrophages activated so would also remove otherparticles from human and animal blood circulation, thus having abeneficial effect on, for instance, allergic reactions.

It is theoretically predictable that the peptides and their fragments,according to the present invention, will be useful for amelioration ofallergic reactions, because the mentioned peptides activate T cells.Allergic reactions are mediated by activated B cells as well asinterleukin-4-secreting T (T_(H2)) cells; this cytokine is necessary forIgE production by B cells. There are literature data concerning role ofβ-endorphin in the regulation of cytokine production by T cells:β-endorphin stimulates IL-2, IL-4 and Υ-interferon production by murineCD4⁺ T cells (van den Bergh P. et al., 1994, Cell. Immunol. 154 109-122;van den Bergh P. et al., 1994, Lymphokine Cytokine Res., 13, 63-69). Itwas shown that β-endorphin could modulate the magnitude of an IL-4induced IgE response (Aebisher I. 1996, Exp. Dermatol., 5, 38-44).

The present invention shows that pentarphins and certain other peptideshave sequence homologies (see FIG. 1). All these peptides have strongphysiological effects on animal and human organisms. According to thisinvention, at least macrophages and T-lymphocytes have receptors forpentarphins. Both of these cells are crucial for organism's resistanceagainst microbial attacks. T-cell activation takes place at the firststage of the development of specific immune response to infection.Antigen-primed naive T cells differentiate into either helper cells(T_(H2)) or inflammatory cells (T_(H1)). T_(H2) cells participate in thedevelopment of humoral immunity (production of specific antibodies) andT_(H1) cells take place in macrophage activation. Immunorphin and itsfragments are not species-specific, since our results show that thesepeptides bind with high affinity to specific receptors on both mouse andhuman cells. Immunorphin is a fragment of the constant part of the heavychain of IgG. This fragment has the same sequence at least in human,mouse, and rat IgG.

Numerous methods of preparing drug formulations in the context ofpeptide drugs, including tuftsin, have been described in literature(see, for example, U.S. Pat. No. 4,816,560, EP0448811, EP0253190). Hightolerance of cyclopentarphin to the action of proteolytic enzymesenables its administration not only intramuscularly but also per os andin nosal. According to the present invention, it is possible to find outappropriate formulations using pentarphins alone as effectivesubstances, or combinations of pentaprphins with antibiotics, such aspenicillins, cefalosporins, tetracyclins, streptomycins, laevomycetins,polymyxins, rifamycins, peptide antibiotics (exemplified by gramicidins,bacitracins, and polymycsins).

It is evident that pentarphins structure (5-residue peptide, minimal)forms the very basis of the present invention. With the knowledgedescribed in this invention, it is possible to design many other activestructures. For example, it is possible to create polymeric forms ofpentarphins with or without using synthetic or biological linkermolecules between the active units. Further, it is possible to addflanking sequences outside of N- and C-terminal ends, or to add suchones to the amino acid side chains (to the Lys residue) to changesolubility etc. It is also possible to prepare macromolecular carrierswith one or more active peptides linked to them.

In the following the invention is further illustrated by non-limitingexamples.

EXAMPLE 1 Pentarphins and Immunorphins Activate Cells of Immune System

(3-[¹²⁵I]iodotyrosyl²⁷)β-endorphin (˜2000 Ci/mmol specific activity) waspurchased from Amersham (UK). Na¹²⁵I (2×10⁶ Ci/M specific activity) wasfrom Russian Scientific Center “Applied Chemistry” (St. Petersburg,Russia). All media, sera for culturing cells, 1,3,4,6-tetra-chloro-3α,6αdiphenyl glycoluril (Iodogen) and other chemicals were obtained fromSigma (St. Louis, Mo.). The decapeptide encompassing the sequence364-373 of HuIgG (immunorphin) and its fragments were synthesized byusing pentafluorophenyl ethers of N-protected amino acids. The peptideswere purified by HPLC followed by fast atom bombardment massspectrometric analysis. Only peptides that were >95% pure were used.

Mononuclear cells were separated from healthy donors blood according tothe Boyum method. T lymphocytes were purified by a nylon wool columnfiltration method. The non-adherent fraction was eluted with RPMI-1640medium supplemented with 5% heat-inactivated fetal calf serum. Thisfraction contained T lymphocytes and small amounts of monocytes and Blymphocytes.

The binding of [¹²⁵I]β-endorphin to T lymphocytes was measured asfollows: 10⁶ cells per tube were incubated with labeled peptide at aconcentration of 10⁻⁷-10⁻¹¹ M for 1 h at 4° C. in 1 ml RPMI-1640 mediumcontaining 20 mM NaN₃ and 10 mM Hepes, pH 7.5. The incubation wasterminated by rapid filtration through GF/A glass fiber filters(Whatman, UK) under vacuum pressure. Filters were rinsed twice with 5 mlvolumes of ice-cold 0.15 M NaCl. The cell-bound radioactivity wasmeasured using ¹²¹I Minigamma counter (LKB, Sweden). Non-specificbinding of [¹²⁵I]β-endorphin was measured in the presence of 10 μMunlabeled β-endorphin. The equilibrium dissociation constant (K_(d)) wasestimated by Scatchard analysis.

Immunorphin (10 μg) and the peptide H-VKGFY-OH (SEQ ID NO:4) (10 μg)were labeled by solid phase oxidation method using Iodogen and Na¹²⁵I (1mCi). The labeled peptides were purified by gel filtration on SephadexG-10 (0.9×10 cm column, 0.05 M phosphate buffer, pH 7.5). The purity ofthe labeled peptides was tested by thin-layer chromatography onaluminium oxide glass with n-butano/acetic acid/water (4:1:1) solventsystem, followed by autoradiography. The specific activity of[¹²⁵I]-immunorphin and [¹²⁵I]-H-VKGFY-OH were 232 Ci/mmol and 179Ci/mmol respectively. Assay of [¹²⁵I]-immunorphin (10⁻¹⁰-10⁻⁷ M) and[¹²⁵I]-H-VKGFY-OH (10⁻¹⁰-10⁻⁷ M) binding to T lymphocytes (10⁶ cells pertube) was carried out in 1 ml RPMI-1640 medium containing 10 mM Hepes,20 mM NaN₃, and 0.6 mg/L PMSF, pH 7.4 at 4° C. for 40 min. The reactionmixture was then filtered through GF/A filters (Whatman, UK). Filterswere rinsed twice with 5 ml volumes of ice-cold 0.15 M NaCl, pH 7.4.Radioactivity was counted using 1211 Minigamma counter (LKB).Non-specific binding of the labeled peptide was measured in the presenceof 10 μM of the unlabeled peptide.

To test the inhibitory effect of unlabeled naloxone, Met-enkephalin, andimmunorphin fragments on the binding of [¹²⁵I]β-endorphin, T lymphocytes(10⁶ cells per tube) were incubated with 1 nM [¹²⁵I]β-endorphin andunlabeled ligands at various concentrations (10⁻¹⁰-10⁻⁶ M) as describedin Section 2.4. The results were plotted as percentage of specificbinding vs. log of competitor concentration, and IC₅₀ values weredetermined graphically. The inhibition constant (K_(i)) was calculatedaccording to the equation:K _(i) =IC ₅₀/(1+[L]/K _(d)),where [L] is a molar concentration of [¹²⁵I]β-endorphin, K_(d) is thedissociation constant of [¹²⁵I]β-endorphin/receptor complex, and IC₅₀ isthe concentration of the competing ligand causing half-maximumdisplacement of [¹²⁵I]β-endorphin. β-Endorphin was found to interactspecifically with T lymphocytes separated from normal human blood. Thedata propose the presence of one class binding sites with the K_(d)value of 0.25±0.03 nM. Non-specific [¹²⁵I]β-endorphin binding thatoccurs in the presence of 10 μM unlabeled β-endorphin constitutes about11% of the total binding value.

To examine the specificity of the β-endorphin binding sites, competitionexperiments were performed using the constant concentration of[¹²⁵I]β-endorphin and increasing concentrations of unlabeled ligands(naloxone, Met-enkephalin, immunorphin and eight synthetic immunophinfragments with various chain lengths; see section 2.5 of the previouspart). Displacement curves indicated that only six unlabelled peptides(H-VKGFY-OH (SEQ ID NO:4), H-LVKGFY-OH (SEQ ID NO:5), H-CLVKGFY-OH (SEQID NO:6), H-TCLVKGFY-OH (SEQ ID NO:7), H-LTCLVKGFY-OH (SEQ ID NO:8) andimmunorphin) were able to compete with [¹²⁵I]β-endorphin for the samebinding sites. The K_(i) values correlate with ligand-receptor affinityand inhibiting potential of ligands. The results of the displacementassay demonstrated that β-endorphin binding to this type of receptors isnot inhibited by naloxone and Met-enkephalin. A minimum fragment ofimmunorphin retaining its inhibitory activity in the competition testwas found to be the pentapeptide H-VKGFY—OH (SEQ ID NO:4). Thepentapeptide was characterized by lesser inhibiting capacity (K_(i)=15nM) as compared to immunorphin (0.6 nM) and its longer fragments(H-LVKGFY-OH (SEQ ID NO:5), K_(i)=8.0 nM; H-CLVKGFY-OH (SEQ ID NO:6),K_(i)=3.4 nM; H-TCLVKGFY-OH (SEQ ID NO:7), K_(i)=2.2 nM; H-LTCLVKGFY-OH(SEQ ID NO:8, K_(i)=1.0 nM). Thus, the β-endorphin receptors expressedby T lymphocytes are highly specific and naloxone-insensitive ones.

The Scatchard plots demonstrate the specific binding of[¹²⁵1]-immunorphin to T lymphocytes in the absence (plot 1;K_(d)=7.0±0.3 nM) and in the presence (plot 2; K_(d)=7.4±0.2 nM) ofnaloxone. The results of this experiment confirmed that naloxone doesnot influence the kinetic of [¹²⁵I]-immunorphin binding to the receptorson T lymphocytes.

The displacement assays demonstrated that pentapeptide H-VKGFY-OH (SEQID NO:4) was the shortest active immunorphin fragment. We prepared[¹²⁵I]-H-VKGFY-OH and studied its interaction with T lymphocytes.Scatchard analysis of the binding showed that the data best fit aone-site model. The K_(d) value for [¹²⁵I]-H-VKGFY-OH/receptor complexwas 36.3±0.5 nM. Non-specific binding of the labeled peptide to Tlymphocytes was about 8% of its total binding to these cells.

The results of the binding assays confirmed that lymphocytes separatedfrom normal human blood express high affinity binding sites forβ-endorphin, K_(d)=(0.25±0.03) nM. The binding of β-endorphin to thesesites was naloxone- and Met-enkephalin-insensitive, but sensitive toimmunorphin and its fragments H-VKGFY-OH (SEQ ID NO:4), H-LVKGFY-OH (SEQID NO:5), H-CLVKGFY-OH (SEQ ID NO:6), H-TCLVKGFY-OH (SEQ ID NO:7),H-LTCLVKGFY-OH (SEQ ID NO:8) (Tab. 1). Thus, T lymphocytes from normalhuman blood express non-opioid receptors for β-endorphin.

Immunorphin (H-SLTCLVKGFY-OH(SEQ ID NO:3)) is homologous (50%) to theβ-endorphin fragment 10⁻¹⁹ (SQTPLVTLFK)(SEQ ID NO:9). The high affinitybinding of immunorphin (K_(d)=7.0±0.3 nM) and its fragmentH-VKGFY-OH(SEQ ID NO:4) (K_(d)=36.3±0.5 nM) to non-opioid receptors forβ-endorphin on T lymphocytes shows that α-endorphin is a peptide with adualistic nature: its C-terminal moiety binds to non-opiod receptors,whereas its N-terminal enkephalin sequence is responsible forβ-endorphin binding to opioid receptors. Therefore, pentarphins can havea special function in organisms which is related to certain functions ofβ-endorphin. We have found that β-endorphin, immunorphin and pentarphinstimulate Con A-induced proliferation of T lymphocytes from the blood ofhealthy donors. [Met⁵]enkephalin and an antagonist of opioid receptorsnaloxone, tested in parallell, were not active. The stimulating effectof β-endorphin, immunorphin and pentarphin on T lymphocyte proliferationwas not inhibited by naloxone. Thus, these peptides bind to commonnaloxone-insensitive binding sites on T lymphocytes and enhance ConA-induced proliferation of these cells.

EXAMPLE 2 Phagocytosis of S. typhimurium by Macrophages in the Absenceor in the

Presence of Pentarphin, Cyclopentarphin, and Tuftsin

Pentarphin was obtained by a solid-phase synthesis. The crude productwas purified by HPLC on a Zorbax ODS column (4×150 mm, 5 μm particlesize) using linear gradient of water acetonitrile (95%) in 0.2% TCA(10-25%, 20 min) at a flow rate of 1 ml/min. According to the absorbanceat 220 nm, the main substance (pentarphin) content was 98%. The peptidestructure was confirmed by an amino acid analysis under standardconditions with a D500 amino acid analyzer (Durrum, USA). Molecular massof pentarphin was estimated by mass spectrometric analysis using Vision2000 spectrometer (“Thermo Bioanalysis”, Great Britain).

Cyclopentarphin was obtained by the cyclization reaction of linearpentarphin (having the side chain groups protected) through amino groupof Val to an activated carboxyl group of Tyr. The reaction product wasunmasked from protective groups and purified by HPLC as described above.The molar yield was 15%. Mass spectrometric analysis showed themolecular peak of cyclopentarphin (594 Da) in the spectrum.

Peritoneal macrophages were isolated from CBA mice (16-18 g). Thevirulent strain Salmonella typhimurium 415 with typical morphologicaland functional properties was used. LD₅₀ was approximately 100 microbialcells injected intraperitoneally into white mice. S. typhimurium wasgrown in Hottinger's broth for 4-6 h at 37° C., then transferred tobeef-extract agar and incubated at 37° C. for 18 h. Macrophagemonolayers on cover glasses were cultivated in sterile test tubes in 199medium supplemented with streptomycin and penicillin (100 μg/ml each)and inactivated fetal calf serum (5%) at 37° C. 24 h later macrophageswere infected with 199 medium supplemented with serum and S. typhimurium415 (108 microbial cells/ml final concentration). Microorganisms andpeptides (tuftsin, pentarphin, or cyclopentarphin) at particularconcentrations were added to the cultivation medium simultaneously. In 2h the contact between microbes and macrophages was interrupted byreplacing the infection medium with a fresh one supplemented withantibiotics. To prevent the recapture of bacteria released fromdestroyed phagocytes by other cells, cultivation medium was replacedwith a fresh one every two hours. Macrophages on cover glasses (intriplicate for every time point, namely 1,2,4,7 and 12 h) were fixed inmethanol for 7 min. After that, the preparations were stained with 0.1%azur II-eosin water solution for 5 min. Cells (300 per cover glass) wereexamined using light microscope and analyzed for following parameters:phagocytic activity (PA)—a percentage of macrophages, participating inphagocytosis; bacterial cytocidal activity (BCA)—a percentage ofphagocytes, destroyed by intracellular bacteria; and phagocytic number(PN)—an average number of microbes per macrophage.

The values of the main characteristics (PA, PN, BCA) of S. typhimuriumphagocytosis in the absence (control) or in the presence of pentarphin(1 nM), cyclopentarphin (1 nM) and tuftsin (100 nM) are shown inTable 1. BCA in control was more than 65% within 7h, and within 12 h allmacrophage monolayer was destroyed by intracellular microorganisms. Thepresence of 1 nM pentarphin or cyclopentarphin in cultivation mediumresulted in a significant increase in bactericidal activity ofmacrophages. In the presence of pentarphin, phagocytes completelydigested the captured microbes within 12 h, and in the presence ofcyclopentarphin—within 7 h of phagocytosis. Tuftsin acted the same wayat a concentration of 100 nM, that is, its activity was 100 times lower.Thus, pentarphin enhances the bactericidal activity of peritonealmacrophages in relation to S. typhimurium 415, in its presence at aconcentration of 1 nM, phagocytosis of this bacteria in vitro completeswith total digestion of captured microbes. Cyclopentarphin is preferablein a drug composition, because its half-life in biological fluids isconsiderably longer than its linear analogs, as known from prior art.

EXAMPLE 3 Combined Action of Pentarphin and Streptomycin

Macrophage monolayers on cover glasses were cultivated and infected asdescribed above in Example 1. Microorganisms, streptomycine and peptides(pentarphin, or cyclopentarphin) at particular concentrations were addedto the cultivation medium simultaneously. In 2 h, the contact betweenmicrobes and macrophages was interrupted by replacing the infectionmedium with a fresh one supplemented with antibiotics. Macrophages oncover glasses (in triplicate for every time point, namely 1, 2, and 4 h)were fixed in methanol for 7 min. After that, the preparations werestained with 0.1% azur II-eosin water solution for 5 min. Cells (300 percover glass) were examined under a light microscope and analyzed forfollowing parameters: phagocytic activity (PA)—a percentage ofmacrophages, participating in phagocytosis; bacterial cytocidal activity(BCA)—a percentage of phagocytes, destroyed by intracellular bacteria;and phagocytic number (PN)—an average number of microbes per macrophage.The data given in Table 2 show that the combined action of streptomycine(10 μg/ml)

pentarphin (1 nM) allows the antibiotic dose to be lowered 5 times.

EXAMPLE 4 Toxicity and Immunogenicity of Pentarphin and Immunorphin

The toxicity of pentarphin and immunorphin was estimated byintraperitoneal injection of peptides (10, 100, 250, 1000, 2500, and3000 mg/kg of body weight) to BCA mice (16-18g). The LD₅₀ value was 2500mg/kg for each peptide, i.e. this dose is not physiological. Activeconcentrations of both peptides are 10-100 μg/kg. Very low toxicity ofthe peptides can be attributed to the fact that aminoacids are the onlydegradation products of these compounds.

No immunogenicity was observed during several experiments withpentarphin and immunorphin utilizing mice as model animals. This was anexpectable result, because the related peptides, Met-enkephalin andLeu-enkephalin, are known to be nonimmunogenic. In general, smallpeptides do not induce antibody production.

TABLE 1 Effect of cyclopentarphin, pentarphin and tuftsin on digestionof the virulent bacterial strain S. typhimurium 415 by mouse peritonealmacrophages in vitro. Phago- cytosis *PA, **BCA, ***PN, Peptide time, h% ± SEM % ± SEM n ± SEM Control 1 65.33 ± 1.11 1.33 ± 0.67 3.90 ± 0.13 272.67 ± 1.07 13.67 ± 0.69  10.17 ± 0.18  4 62.33 ± 1.29 35.67 ± 1.41 11.17 ± 1.03  7 29.00 ± 0.89 66.67 ± 0.62  15.50 ± 0.34  12 0 100 —Pentarphin 1 84.08 ± 2.12 1.08 ± 0.71 7.11 ± 0.15 (1 nM) 2 89.15 ± 1.781.55 ± 0.65 9.29 ± 0.74 4 66.82 ± 1.14 9.32 ± 1.32 6.02 ± 0.66 7 23.31 ±0.84 10.24. ± 1.17   1.03 ± 0.24 12  3.73 ± 0.64 2.35 ± 1.21 0.66 ± 0.12Cyclo- 1 87.81 ± 3.14 1.25 ± 0.64 7.78 ± 0.39 pentarphin 2  91.33 ±2..19 1.06 ± 0.49 8.81 ± 0.27 (1 nM) 4 57.43 ± 2.01 2.13 ± 0.60 5.32 ±0.51 7  1.74 ± 0.44 2.22 ± 1.31 0.27 ± 0.20 Tuftsin 1 66.07 ± 1.82 1.41± 0.27 5.84 ± 0.27 (100 nM) 2 74.11 ± 1.42 8.12 ± 0.99 7.31 ± 0.25 461.97 ± 1.18 29.34 ± 0.75  6.29 ± 0.48 7 38.48 ± 0.23 31.46 ± 1.23  3.32± 0.56 12  9.31 ± 0.29 7.64 ± 1.38 1.36 ± 0.86 *PA—phagocytic activity,a percentage of macrophages, participating in phagocytosis;**BCA—bacterial cytocidal activity, a percentage of phagocytes,destroyed by intracellular bacteria; ***PN—phagocytic number, an averagenumber of microbes per macrophage.

TABLE 2 Effect of streptomycine + pentarphin on the digestion of thevirulent bacterial strain S. typhimurium 415 by mouse peritonealmacrophages in vitro. Phagocytosis PA, BCA, PN, Peptide time, h % ± SEM% ± SEM n ± SEM Streptomycine 1 86.45 ± 2.13 1.04 ± 0.32 8.56 ± 0.76 (50μg/ml) 2 96.17 ± 1.76 1.12 ± 0.74 2.03 ± 0.46 4 0 0 0 Streptomycine 187.65 ± 2.19 0.94 ± 0.49 8.60 ± 0.58 (10 μg/ml) + 2 98.22 ± 1.94 1.05 ±0.54 2.11 ± 0.44 pentarphin 4 0 0 0 (1 nM) Streptomycine 1 91.66 ± 2.270.99 ± 0.56 8.92 ± 0.83 (10 μg/ml) + 2 99.31 ± 2.44 1.17 ± 0.61 2.01 ±0.39 cyclo- 4 0 0 0 pentarphin (1 nM)

1. A pentarphin molecule consisting of a structure of cyclo(Val-Lys-Gly-Phe-Tyr) and further being called as cyclopentarphin.
 2. Adrug composition comprising pentarphin molecule in a cyclic form andsaid pentarphin molecule consisting of the amino acid sequence accordingto SEQ ID NO:
 4. 3. The drug composition according to claim 2, whereinthe composition additionally contains at least one conventionalantibiotic.
 4. The drug composition according to claim 3, wherein theconventional antibiotic is streptomycin.
 5. A cell cultivation mediumfor mammal cell cultivation, said cultivation further containingmacrophages, said cultivation medium further comprising pentarphinmolecules consisting of the amino acid sequence according to SEQ ID NO:4 and having a cyclic structure at concentrations less than 10 mg/l. 6.The cell cultivation medium according to claim 5, wherein the mediumadditionally contains antibiotics.